BUSCADOR RECOLECTA

The use of more salt stress-tolerant vine rootstocks can be a sustainable strategy for adapting traditional grapevine cultivars to future conditions. However, how the new M1 and M4 rootstocks perform against salinity compared to conventional ones, such as the 1103-Paulsen, had not been previously assessed under real field conditions. Therefore, a field trial was carried out in a young ‘Tempranillo’ (Vitis vinifera L.) vineyard grafted onto all three rootstocks under a semi-arid and hot-summer Mediterranean climate. The vines were irrigated with two kinds of water: a non-saline Control with EC of 0.8 dS m–1 and a Saline treatment with 3.5 dS m–1. Then, various physiological parameters were assessed in the scion, and, additionally, gene expression was studied by high throughput sequencing in leaf and berry tissues. Plant water relations evidenced the osmotic effect of water quality, but not that of the rootstock. Accordingly, leaf-level gas exchange rates were also reduced in all three rootstocks, with M1 inducing significantly lower net photosynthesis rates than 1103-Paulsen. Nevertheless, the expression of groups of genes involved in photosynthesis and amino acid metabolism pathways were not significantly and differentially expressed. The irrigation with saline water significantly increased leaf chloride contents in the scion onto the M-rootstocks, but not onto the 1103P. The limitation for leaf Cl– and Na+ accumulation on the scion was conferred by rootstock. Few processes were differentially regulated in the scion in response to the saline treatment, mainly, in the groups of genes involved in the flavonoids and phenylpropanoids metabolic pathways. However, these transcriptomic effects were not fully reflected in grape phenolic ripeness, with M4 being the only one that did not cause reductions in these compounds in response to salinity, and 1103-Paulsen having the highest overall concentrations. These results suggest that all three rootstocks confer short-term salinity tolerance to the scion. The lower transcriptomic changes and the lower accumulation of potentially phytotoxic ions in the scion grafted onto 1103-Paulsen compared to M-rootstocks point to the former being able to maintain this physiological response in the longer term. Further agronomic trials should be conducted to confirm these effects on vine physiology and transcriptomics in mature vineyards., Peer reviewed

Supplemental table S1: Primers and probes used for grapevine gene expression analysis., The use of more salt stress-tolerant vine rootstocks can be a sustainable strategy for adapting traditional grapevine cultivars to future conditions. However, how the new M1 and M4 rootstocks perform against salinity compared to conventional ones, such as the 1103-Paulsen, had not been previously assessed under real field conditions. Therefore, a field trial was carried out in a young ‘Tempranillo’ (Vitis vinifera L.) vineyard grafted onto all three rootstocks under a semi-arid and hot-summer Mediterranean climate. The vines were irrigated with two kinds of water: a non-saline Control with EC of 0.8 dS m–1 and a Saline treatment with 3.5 dS m–1. Then, various physiological parameters were assessed in the scion, and, additionally, gene expression was studied by high throughput sequencing in leaf and berry tissues. Plant water relations evidenced the osmotic effect of water quality, but not that of the rootstock. Accordingly, leaf-level gas exchange rates were also reduced in all three rootstocks, with M1 inducing significantly lower net photosynthesis rates than 1103-Paulsen. Nevertheless, the expression of groups of genes involved in photosynthesis and amino acid metabolism pathways were not significantly and differentially expressed. The irrigation with saline water significantly increased leaf chloride contents in the scion onto the M-rootstocks, but not onto the 1103P. The limitation for leaf Cl– and Na+ accumulation on the scion was conferred by rootstock. Few processes were differentially regulated in the scion in response to the saline treatment, mainly, in the groups of genes involved in the flavonoids and phenylpropanoids metabolic pathways. However, these transcriptomic effects were not fully reflected in grape phenolic ripeness, with M4 being the only one that did not cause reductions in these compounds in response to salinity, and 1103-Paulsen having the highest overall concentrations. These results suggest that all three rootstocks confer short-term salinity tolerance to the scion. The lower transcriptomic changes and the lower accumulation of potentially phytotoxic ions in the scion grafted onto 1103-Paulsen compared to M-rootstocks point to the former being able to maintain this physiological response in the longer term. Further agronomic trials should be conducted to confirm these effects on vine physiology and transcriptomics in mature vineyards., Peer reviewed

Supplementary Tables Supplementary Table 1: Clinical data of the study cohort. Supplementary Table 2: Functional enrichment by trend of soluble proteins for Diagnostic group study. Supplementary Table 3: Summary of significant soluble proteins for each study after classical statistical analysis. Supplementary Table 4: Functional enrichment of significant soluble proteins for comparison Monoclonal B-cell lymphocytosis (MBLhi) vs. Chronic Lymphocytic Leukemia (CLL) and CLL in progression (p-CLL) vs. Stable/constant CLL (c-CLL) vs. MBLhi and combinations. Supplementary Table 5: Functional enrichment of significant soluble proteins according to trend for Diagnostic group study. Supplementary Table 6: Protein correlation. Supplementary Table 7: Top 20/30 proteins after random forest analyses. Supplementary Table 8: Summary of significant soluble proteins for each study after linear model. Supplementary Table 9: Functional enrichment by trend of soluble proteins for Diagnostic group and treatment line. Supplementary Table 10: Functional enrichment of significant soluble proteins for all combination combinations between Monoclonal B-cell lymphocytosis (MBLhi), Stable/constant Chronic Lymphocytic Leukemia (c-CLL), CLL in progression previously to 1st line treatment (CLL-PFT) and CLL in progression to time from 1st line treatment (CLL-TFT). Supplementary Table 11: Functional enrichment by trend of soluble proteins for Immunoglobulin Heavy chain Variable (IGHV) gene status study. Supplementary Table 12: Functional enrichment of significant soluble proteins for Immunoglobulin Heavy chain Variable (IGHV) gene status study., Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm characterized by the accumulation of mature B cells. The diagnosis is established by the detection of monoclonal B lymphocytes in peripheral blood, even in early stages [monoclonal B-cell lymphocytosis (MBLhi)], and its clinical course is highly heterogeneous. In fact, there are well-characterized multiple prognostic factors that are also related to the observed genetic heterogenicity, such as immunoglobulin heavy chain variable region (IGHV) mutational status, del17p, and TP53 mutations, among others. Moreover, a dysregulation of the immune system (innate and adaptive immunity) has been observed in CLL patients, with strong impact on immune surveillance and consequently on the onset, evolution, and therapy response. In addition, the tumor microenvironment is highly complex and heterogeneous (i.e., matrix, fibroblast, endothelial cells, and immune cells), playing a critical role in the evolution of CLL. In this study, a quantitative profile of 103 proteins (cytokines, chemokines, growth/regulatory factors, immune checkpoints, and soluble receptors) in 67 serum samples (57 CLL and 10 MBLhi) has been systematically evaluated. Also, differential profiles of soluble immune factors that discriminate between MBLhi and CLL (sCD47, sCD27, sTIMD-4, sIL-2R, and sULBP-1), disease progression (sCD48, sCD27, sArginase-1, sLAG-3, IL-4, and sIL-2R), or among profiles correlated with other prognostic factors, such as IGHV mutational status (CXCL11/I-TAC, CXCL10/IP-10, sHEVM, and sLAG-3), were deciphered. These results pave the way to explore the role of soluble immune checkpoints as a promising source of biomarkers in CLL, to provide novel insights into the immune suppression process and/or dysfunction, mostly on T cells, in combination with cellular balance disruption and microenvironment polarization leading to tumor escape., Peer reviewed

The use of more salt stress-tolerant vine rootstocks can be a sustainable strategy for adapting traditional grapevine cultivars to future conditions. However, how the new M1 and M4 rootstocks perform against salinity compared to conventional ones, such as the 1103-Paulsen, had not been previously assessed under real field conditions. Therefore, a field trial was carried out in a young ‘Tempranillo’ (Vitis vinifera L.) vineyard grafted onto all three rootstocks under a semi-arid and hot-summer Mediterranean climate. The vines were irrigated with two kinds of water: a non-saline Control with EC of 0.8 dS m–1 and a Saline treatment with 3.5 dS m–1. Then, various physiological parameters were assessed in the scion, and, additionally, gene expression was studied by high throughput sequencing in leaf and berry tissues. Plant water relations evidenced the osmotic effect of water quality, but not that of the rootstock. Accordingly, leaf-level gas exchange rates were also reduced in all three rootstocks, with M1 inducing significantly lower net photosynthesis rates than 1103-Paulsen. Nevertheless, the expression of groups of genes involved in photosynthesis and amino acid metabolism pathways were not significantly and differentially expressed. The irrigation with saline water significantly increased leaf chloride contents in the scion onto the M-rootstocks, but not onto the 1103P. The limitation for leaf Cl– and Na+ accumulation on the scion was conferred by rootstock. Few processes were differentially regulated in the scion in response to the saline treatment, mainly, in the groups of genes involved in the flavonoids and phenylpropanoids metabolic pathways. However, these transcriptomic effects were not fully reflected in grape phenolic ripeness, with M4 being the only one that did not cause reductions in these compounds in response to salinity, and 1103-Paulsen having the highest overall concentrations. These results suggest that all three rootstocks confer short-term salinity tolerance to the scion. The lower transcriptomic changes and the lower accumulation of potentially phytotoxic ions in the scion grafted onto 1103-Paulsen compared to M-rootstocks point to the former being able to maintain this physiological response in the longer term. Further agronomic trials should be conducted to confirm these effects on vine physiology and transcriptomics in mature vineyards., Peer reviewed

Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm characterized by the accumulation of mature B cells. The diagnosis is established by the detection of monoclonal B lymphocytes in peripheral blood, even in early stages [monoclonal B-cell lymphocytosis (MBLhi)], and its clinical course is highly heterogeneous. In fact, there are well-characterized multiple prognostic factors that are also related to the observed genetic heterogenicity, such as immunoglobulin heavy chain variable region (IGHV) mutational status, del17p, and TP53 mutations, among others. Moreover, a dysregulation of the immune system (innate and adaptive immunity) has been observed in CLL patients, with strong impact on immune surveillance and consequently on the onset, evolution, and therapy response. In addition, the tumor microenvironment is highly complex and heterogeneous (i.e., matrix, fibroblast, endothelial cells, and immune cells), playing a critical role in the evolution of CLL. In this study, a quantitative profile of 103 proteins (cytokines, chemokines, growth/regulatory factors, immune checkpoints, and soluble receptors) in 67 serum samples (57 CLL and 10 MBLhi) has been systematically evaluated. Also, differential profiles of soluble immune factors that discriminate between MBLhi and CLL (sCD47, sCD27, sTIMD-4, sIL-2R, and sULBP-1), disease progression (sCD48, sCD27, sArginase-1, sLAG-3, IL-4, and sIL-2R), or among profiles correlated with other prognostic factors, such as IGHV mutational status (CXCL11/I-TAC, CXCL10/IP-10, sHEVM, and sLAG-3), were deciphered. These results pave the way to explore the role of soluble immune checkpoints as a promising source of biomarkers in CLL, to provide novel insights into the immune suppression process and/or dysfunction, mostly on T cells, in combination with cellular balance disruption and microenvironment polarization leading to tumor escape., Peer reviewed

Gluten proteins are responsible for the unique viscoelastic properties of wheat dough, but they also trigger the immune response in celiac disease patients. RNA interference (RNAi) wheat lines with strongly silenced gliadins were obtained to reduce the immunogenic response of wheat. The E82 line presents the highest reduction of gluten, but other grain proteins increased, maintaining a total nitrogen content comparable to that of the wild type. To better understand the regulatory mechanisms in response to gliadin silencing, we carried out a transcriptomic analysis of grain and leaf tissues of the E82 line during grain filling. A network of candidate transcription factors (TFs) that regulates the synthesis of the seed storage proteins (SSPs), α-amylase/trypsin inhibitors, lipid transfer proteins, serpins, and starch in the grain was obtained. Moreover, there were a high number of differentially expressed genes in the leaf of E82, where processes such as nutrient availability and transport were enriched. The source-sink communication between leaf and grain showed that many down-regulated genes were related to protease activity, amino acid and sugar metabolism, and their transport. In the leaf, specific proline transporters and lysine-histidine transporters were down- and up-regulated, respectively. Overall, the silencing of gliadins in the RNAi line is compensated mainly with lysine-rich globulins, which are not related to the proposed candidate network of TFs, suggesting that these proteins are regulated independently of the other SSPs. Results reported here can explain the protein compensation mechanisms and contribute to decipher the complex TF network operating during grain filling., Peer reviewed

Supplementary Table 8. Top 3 Gene Ontology (GO) terms enriched for each candidate transcription factor (TF) summarized from previously published GO enrichment analysis for GENIE3 network genes in Ramírez-González et al., 2018. The black boxes indicate that this is an enriched GO for this TF gene., Gluten proteins are responsible for the unique viscoelastic properties of wheat dough, but they also trigger the immune response in celiac disease patients. RNA interference (RNAi) wheat lines with strongly silenced gliadins were obtained to reduce the immunogenic response of wheat. The E82 line presents the highest reduction of gluten, but other grain proteins increased, maintaining a total nitrogen content comparable to that of the wild type. To better understand the regulatory mechanisms in response to gliadin silencing, we carried out a transcriptomic analysis of grain and leaf tissues of the E82 line during grain filling. A network of candidate transcription factors (TFs) that regulates the synthesis of the seed storage proteins (SSPs), α-amylase/trypsin inhibitors, lipid transfer proteins, serpins, and starch in the grain was obtained. Moreover, there were a high number of differentially expressed genes in the leaf of E82, where processes such as nutrient availability and transport were enriched. The source-sink communication between leaf and grain showed that many down-regulated genes were related to protease activity, amino acid and sugar metabolism, and their transport. In the leaf, specific proline transporters and lysine-histidine transporters were down- and up-regulated, respectively. Overall, the silencing of gliadins in the RNAi line is compensated mainly with lysine-rich globulins, which are not related to the proposed candidate network of TFs, suggesting that these proteins are regulated independently of the other SSPs. Results reported here can explain the protein compensation mechanisms and contribute to decipher the complex TF network operating during grain filling., Peer reviewed

Supplementary Table 9. Complete results of Gene Ontology (GO) enrichment analysis of genes in module 13 of co-expression network WGCNA published in Ramírez-González et al. (2018). GO:MF, Molecular Function domain; GO:BP, Biological Process domain; GO:CC, Cellular Component domain., Gluten proteins are responsible for the unique viscoelastic properties of wheat dough, but they also trigger the immune response in celiac disease patients. RNA interference (RNAi) wheat lines with strongly silenced gliadins were obtained to reduce the immunogenic response of wheat. The E82 line presents the highest reduction of gluten, but other grain proteins increased, maintaining a total nitrogen content comparable to that of the wild type. To better understand the regulatory mechanisms in response to gliadin silencing, we carried out a transcriptomic analysis of grain and leaf tissues of the E82 line during grain filling. A network of candidate transcription factors (TFs) that regulates the synthesis of the seed storage proteins (SSPs), α-amylase/trypsin inhibitors, lipid transfer proteins, serpins, and starch in the grain was obtained. Moreover, there were a high number of differentially expressed genes in the leaf of E82, where processes such as nutrient availability and transport were enriched. The source-sink communication between leaf and grain showed that many down-regulated genes were related to protease activity, amino acid and sugar metabolism, and their transport. In the leaf, specific proline transporters and lysine-histidine transporters were down- and up-regulated, respectively. Overall, the silencing of gliadins in the RNAi line is compensated mainly with lysine-rich globulins, which are not related to the proposed candidate network of TFs, suggesting that these proteins are regulated independently of the other SSPs. Results reported here can explain the protein compensation mechanisms and contribute to decipher the complex TF network operating during grain filling., Peer reviewed

Suppl. File 1. List of wild Helianthus accessions used in this study and associated phenotyping assays. Seventy one accessions were phenotyped in 6 L pots: 36 wild H. annuus accessions and 35 wild Helianthus accessions from other species than annuus, including 21 annual accessions from 8 species (12 taxa) and 14 perennial accessions from 7 species. Eighteen of these accessions were phenotyped for the exudate activity on broomrape seed germination and in rhizotrons. Suppl. File 2. Timeline of the culture and phenotyping in rhizotrons. Phenotyping in rhizotrons of the wild Helianthus species and the control cultivated H. annuus was performed at the attachment stage (14 days after inoculation-dai), tubercle stage (21 dai) and necrotic tubercle stage (28 dai). At 14 dai, samples (fragments of roots with compatible or incompatible attachments) were prepared for cytological studies. D0: Day 0 is the day of inoculation. Suppl. File 3. Numbers of rhizotrons and independent experiments/ accessions. Rhizotrons were inoculated with the race E-BOU (2017). At least 2 independent experiments were performed for each accession. The numbers of Non-Treated (NT) and Treated (T) rhizotrons (with GR24 + DCL) were variable depending on the germinating ability of the accession. At 14 dai and 21 dai, the number of attachments and tubercles were counted respectively. As one rhizotron/ experiment was used for cytological sampling at 14 dai, the number of rhizotrons differed between 14 and 21 dai. Suppl. File 4. Raw data of the number of attachments (at 14 dai), tubercles (at 21 dai) and necrotic tubercles (at 28 dai) / rhizotron for each accession. Raw data are detailed in the specific joined file. NA: data Not Available. Wild Helianthus plantlets were inoculated with conditioned Orobanche seeds (race E-BOU), following 15 days of culture in soil and 6 days in rhizotrons (see Materials and Methods and Suppl. File 2) except for the following experiments: I15: 17 days of culture in soil, and inoculation the day of transfer in rhizotron; I16 and I18: inoculation the day of transfer in rhizotron; I19: plantlets were grown 27 days in soil and 6 days in rhizotron before inoculation. Suppl. File 5. Germination dose-response curves of various O. cumana populations to various germination stimulants. Germination dose-response curves were modelled after normalization of the germination activity (bar : ± SD) thanks to the germination percentage of each populations obtained with 1 μM GR24 (Fig. 1a) using a four parameter logistic curve. For each compound, (±)-GR24; DCL and costunolide, and equimolar mixtures, a range of concentrations from 1 μM to 0.1 pM were applied to conditioned seeds of five O. cumana populations. Suppl. File 6. Half-maximal effective concentration (EC50) of various germination stimulants on various O. cumana populations. EC50 was determined for every compounds and mixtures, and for the five O. cumana populations thanks to the generated dose-response curves presented in the Suppl. File 5. Bar : ± SE. Different letters indicate significant differences at p < 0.05 (Student-Newman-Keuls Methodtest) between germination stimulants for a population (corresponding colored letters) or between population for a germination stimulant (black letters). Due to poor germination in response to DCL and costunolide, the EC50 of the G-RO population could not be determined. Suppl. File 7. Kinetic of GS activity exuded by a set of Helianthus accessions. Germination activities were normalized thanks to the germination percentage of both population obtained with GR24:DCL (equimolar, 1μM). For each accessions (except #774a and #826a), root exudates collected 3, 4, 5 and 6 weeks after sowing were applied to conditioned seeds of the two populations, E-BOU and G-RO. For #774a and #826a root exudates were collected at 3 and 4 weeks of culture only. Bar : ± SE. Suppl. File 8. Quantitative analysis of the phenotyping data at early stages (attachments and tubercles) in rhizotrons inoculated with the race E-BOU. Data were analysed taking into account only GS-treated rhizotrons, except for the cultivated susceptible controls XRQ and 2603. 8a. There was no significant statistical difference in the number of attachments at 14 dai between the wild resistant accessions except for the wild H. annuus #826a. 8b. Ordering the accessions by the efficiency of the development of attachments into tubercles (% of tubercle/ attachment) revealed a significant difference between phenotyping classes I and III, compared to the classes II and IV. Suppl. File 9. Quantitative analysis of the percentage of necrotic tubercles at 28 dai in rhizotrons inoculated with the race E-BOU. Data were analysed taking into account only GS-treated rhizotrons, except for the cultivated susceptible controls XRQ and 2603 (non-treated rhizotrons). Accessions without tubercle development were not taken into account (Class I and Class III, except #833a which develop few tubercles). Statistical analysis was performed using the Kruskal-Wallis test (α = 0.05). Suppl. File 10. List of wild Helianthus accessions used for cytological studies and number of observed samples for each accession. A few accessions from each phenotyping class in rhizotrons were used, inoculated with the race E-BOU. Whole root segment with attachment were cleared with chloral hydrate, or sectioned, imbedded in technovit system and stained with toluidine blue O. Attachments of accessions from classes I and III were all Incompatible (IA). Attachments of accessions from class IV were all Compatible (CA). For the accessions from Class II there were a mixture of IA and CA. As accessions from Classes I and III did not induce seed germination, attachments were sampled only from GS-treated rhizotrons for these accessions. Suppl. File 11. Summary of the the cellular phenotypes observed by cytology on attachments (14 dai). Common cellular resistant mechanisms led to incompatible attachments (IA) independently of the phenotyping classes in rhizotrons. Rarely, some defence reactions were observed in compatible attachments (CA). Suppl. File 12. Defence reactions at proximity of compatible attachments revealed by cytological study of Class II accessions. At 14 dai, accessions from class II, such as H. praecox, developed a mixture of incompatible attachments and compatible attachments. In some cases, defence reactions at proximity of these compatible attachments were observed as green staining suggesting phenolic compounds (a. accession #677 H. preacox; arrowheads), or gumlike substance in xylem vessels (b. accesion #679 H. praecox; red asterisks). Bar = 100 μ. Suppl. File 13. Phenotyping in 6 L pots of wild Helianthus using the most virulent broomrape races G. Boxplots of 7 accessions phenotyped in pots following inoculation with 4 races G (assay No 3) The accessions #2601 H. exilis and #325 H. tuberosus were from class I, #584 H. bolanderi, #677 H. praecox, #761 H. petiolaris and #786 H. debilis tardiflorus from class II and #833 H. annuus from class III. The 4 races G were from Romania (G-RO), Russia (G-RU), Spain (G-GV) and Turkey (G-TK). Five to 8 pots were cultured for each accession/ race. In each pot, the number of broomrape emergences was counted at the time of sunflower flowering. For each accession, Kruskal-Wallis test was performed (α=0.05). The p values were respectively: #2601ex: p=0.29; #325t (no p value); #584b: p=0.52; #677pr: p=0.12; #761p: p=0.004; #786d: p=0.03; #833a: p=0.06; B117: p=0.29; LP2:p=0.008., Orobanche cumana Wall., sunflower broomrape, is one of the major pests for the sunflower crop. Breeding for resistant varieties in sunflower has been the most efficient method to control this parasitic weed. However, more virulent broomrape populations continuously emerge by overcoming genetic resistance. It is thus essential to identify new broomrape resistances acting at various stages of the interaction and combine them to improve resistance durability. In this study, 71 wild sunflowers and wild relatives accessions from 16 Helianthus species were screened in pots for their resistance to broomrape at the late emergence stage. From this initial screen, 18 accessions from 9 species showing resistance, were phenotyped at early stages of the interaction: the induction of broomrape seed germination by sunflower root exudates, the attachment to the host root and the development of tubercles in rhizotron assays. We showed that wild Helianthus accessions are an important source of resistance to the most virulent broomrape races, affecting various stages of the interaction: the inability to induce broomrape seed germination, the development of incompatible attachments or necrotic tubercles, and the arrest of emerged structure growth. Cytological studies of incompatible attachments showed that several cellular mechanisms were shared among resistant Helianthus species., Peer reviewed

Orobanche cumana Wall., sunflower broomrape, is one of the major pests for the sunflower crop. Breeding for resistant varieties in sunflower has been the most efficient method to control this parasitic weed. However, more virulent broomrape populations continuously emerge by overcoming genetic resistance. It is thus essential to identify new broomrape resistances acting at various stages of the interaction and combine them to improve resistance durability. In this study, 71 wild sunflowers and wild relatives accessions from 16 Helianthus species were screened in pots for their resistance to broomrape at the late emergence stage. From this initial screen, 18 accessions from 9 species showing resistance, were phenotyped at early stages of the interaction: the induction of broomrape seed germination by sunflower root exudates, the attachment to the host root and the development of tubercles in rhizotron assays. We showed that wild Helianthus accessions are an important source of resistance to the most virulent broomrape races, affecting various stages of the interaction: the inability to induce broomrape seed germination, the development of incompatible attachments or necrotic tubercles, and the arrest of emerged structure growth. Cytological studies of incompatible attachments showed that several cellular mechanisms were shared among resistant Helianthus species., Peer reviewed